Genetic modification of pluripotent hemopoietic stem cells from primates (P-PHSC) has been an elusive goal for many years. Retrovirus vectors have been used in the past with limited success [1]. Though retroviral vector technology is still improving, progress in increasing the transduction of P-PHSC is slow. This is due to the fact that a solution is not straightforward and that the P-PHSC cannot be identified by a rapid in vitro culture method [1]. Though culture of hemopoie-tic progenitor cells is possible, the in vitro transduction levels of these cells do not reflect transduction of P-PHSC that in vivo can grow out to give long term reconstitution in multi-hemopoietic lineages [1,2,3]. Although long-term in vitro culture assays, such as, e.g., the so-called LTC-IC assay, have long been considered relevant assays for P-PHSC, it is now generally accepted that only a very minor sub-population of the cells identified in long-term in vitro culture assays are P-PHSC. Therefore, genetic modification of long-term in vitro cultured cells, even very efficient genetic modification, does not provide any relevant information on genetic modification of P-PHSC. Furthermore, although increasing knowledge is being gathered on the expression of cell surface markers on P-PHSC, P-PHSC can also not be identified by their phenotype. P-PHSC are known to express the CD34 molecule and to be negative for many other hemopoietic cell surface markers, but even the purest P-PHSC population that can currently be phenotypically characterized contains only few P-PHSC. Due to this, transduction has to be evaluated by laborious and lengthy in vivo studies using a bone marrow transplantation setting where the stem cells in the bone marrow were transduced ex vivo and subsequently transplanted back into monkey or human. Transduction of P-PUSC is verified by the long term persistence of genetically modified hemopoietic cells. Currently, the most efficient method for the transduction of P-PHSC is by means of retroviral vectors. Using such vectors, it is possible to transduce approx. up to 0.01-0.1% of the P-PHSC [3,4,5,6,7]. The limitation of retroviral transduction is most likely due to a restricted expression of the retrovirus receptor on P-PHSC, combined with the fact that P-PHSC are usually not in cell cycle, whereas retroviral vectors do not efficiently transduce non-dividing cells [8,9,10,11].
A number of methods have been devised to improve the P-PHSC transduction by retroviral vectors such as pseudotyping retroviruses using VSV (Vesicular Stomatitis Virus) envelope protein or GALV (Gibbon Ape Leukemia Virus) envelope proteins to target different and possibly more abundantly present receptors on the cell membrane. Other strategies were directed toward improving the number of cycling P-PHSC in the transplant. To date, this did not result in-a significant improvement of P-PHSC transduction.
In contrast to P-PHSC, murine PHSC are very easily transduced by the current generation of retroviral vectors. This observation, made in experiments using retroviral vectors, shows that successful gene transfer into murine PHSC is by no means indicative for successful gene transfer into P-PHSC. One can think of a number of different possible reasons for this observation. We hypothesized that it is theoretically not optimal to use a vector system that has evolved in murine animals for humans. Though the cellular processes involved in the murine retrovirus life cycle are conserved between murine mammals and primates, it is very well possible that the evolutionary divergence of the species resulted in structural differences in the related proteins that affect the functional efficiency of the murine virus proteins in human cells and, thus, affect the transduction process. To avoid these problems, we turned to a different vector system based on the human virus adeno-associated virus (AAV).
AAV is a human virus of the parvovirus family. The AAV genome is encapsidated as a linear single-stranded DNA molecule of approximately 5 kb. Both the plus and the minus strand are infectious and are packaged into virions [12,13]. Efficient AAV replication does not occur unless the cell is also infected by adenavirus or herpes virus. In the absence of helper virus, AAV establishes a latent infection in which its genome is integrated into the cellular chromosomal DNA. The AAV genome contains two large open reading frames. The left half of the genome encodes regulatory proteins, termed REP proteins, that govern replication of AAV-DNA during a lytic infection. The right half encodes the virus structural proteins VP1, VP2 and VP3 that together form the capsid of the virus. The protein coding region is flanked by inverted terminal repeats (ITRs) of 145 bp each, which appear to contain all the cis-acting sequences required for virus replication, encapsidation and integration into the host chromosome [14,15].
In an AAV-vector, the entire protein-coding domain (.+-.4.3 kb) can be replaced by the gene(s) of interest, leaving only the flanking ITRs intact. such vectors are packaged into virions by supplying the AAV-proteins in trans. This can be achieved by a number of different methods, one of them encompassing a transfection into adenovirus infected cells of a vector plasmid carrying a sequence of interest flanked by two ITRs and a packaging plasmid carrying the in trans required AAV protein coding domains rep and cap [15,16,17,18,19]. Due to the stability of the AAV-virion, the adenovirus contamination can be cleared from the virus preparation by heat inactivation (1 hr, 56.degree. C.). In initial studies, virus preparations were contaminated with wild-type AAV, presumably due to recombination events between the vector and the helper construct [16,17,18,19]. Currently, wild-type AAV-free recombinant AAV stocks can be generated by using packaging constructs that do not contain any sequence homology with the vector [15].
Several characteristics distinguish AAV-vectors from the classical retroviral vectors (see also table 1). AAV is a DNA virus which means that the gene of interest, within the size-constraints of AAV, can be inserted as a genomic clone [20, 21]. Some genes, most notably the human .beta.-globin gene, require the presence of introns for efficient expression of the gene [22]. Genomic clones of genes cannot be incorporated easily in retroviral vectors, as these will splice out the introris during the RNA-stage of their life-cycle [23].
In human target cells, wild-type AAV integrates, preferentially, into a discrete region (19q13.3-qter) of chromosome 19 [24,25,26]. This activity might correlate with rep-gene expression in the target cell, since it was found that the large rep-proteins bind to, the human integration site in vitro [27]. AAV-vectors do integrate with high efficiency into the host chromosomal DNA, however, thus far, they do not share the integration site specificity of wtAAV [20]. Site-speciftc integration would be of great importance since it reduces the risks of transformation of the target cell through insertional mutagenesis. Wild-type AAV is, thus far, not associated with human disease. Evidence is accumulating that AAV infection of a cell, indeed, forms an extra barrier against its malignant transformation (reviewed in [28]). In contrast to retroviral vectors where, due to the extended packaging signal, parts of the gag-region need to be present in the vector, the entire protein coding domain of AAV can be deleted and replaced by the sequences of interest, thus totally avoiding any inTmunogenicity problem associated with viral protein expression in transduced target cells. One drawback of AAV-vectors is that they are derived from a human virus. Thus, patients treated with an AAV-vector might become exposed to wtAAV which, in the presence of a helper virus such as adeno-virus or herpes simplex virus, can supply the virus replication and packaging proteins in trans and thus induce spread of the recombinant AAV-virus into the environment. This is a feature not shared by the currently used MuLV-derived retroviral vectors; wild-type MuLV's do not normally cause infections in humans. The risk of recombinant AAV spread into the environment must, however, not be overestimated since it requires the presence of wtAAV and a helper virus. This is not a frequently occurring situation. In addition, during the integration process of AAV-vectors, often the ITRs undergo some form of recombination leading to loss of function [15]. Such proviruses cannot be rescued and, thus, provide an additional safety level of these vectors.
The first AAV-vectors were made by replacing part of the AAV-coding region with either the Chloramphenicol Acetyl-transferase (CAT) or the neon gene [16,17]. All of these vectors retained either a functional rep- or a functional cap-coding region. Recombinant virus was generated by cotransfection with a plasmid containing a complete AAV-genome. The recombinant AAV-CAT virus conferred Chloramphenicol Acetyltransferase activity to 293 cells [16] whereas the recombinant neo.sup.R virus conferred G418-resistance to Human Detroit 6 cells, KB-cells and mouse L-cells [71].
Currently, AAV-vectors are made that are totally devoid of AAV-protein coding sequences. Typically, virus is made from these vectors by complementation with a plasmid carrying the AAV-protein coding region but no ITR-sequences [15].
AAV-vector technology is under development for a number of different therapeutic purposes and target tissues. The as yet most developed system is, perhaps, AAV-vector mediated gene transfer to lung cells [29,30]. AAV-vectors carrying the neo.sup.R gene or the CAT gene were transferred and expressed efficiently in airway epithelial cells [29]. An AAV-vector carrying sequences 486-4629 of the human Cystic Fibrosis Transmembrane conductance Regulator (CFTR) gene fused to a synthetic oligonucleotide supplying the translation start site, was capable of complementing Cystic fibrosis (CF) in vitro [31]. In addition, stable gene transfer and expression was reported following infection of primary CF nasal polyp cells and after in vivo delivery of the AAV-CVTR vector to one lobe of the rabbit lung [30]. In vivo, the vector DNA could be detected in 50% of the nuclei at 3 months post-administration. Although the prevalence of the vector decreased after this time point, still .+-.5% of the nuclei were positive at the six months time point [30]. The presence of the vector correlated well with expression of RNA and recombinant protein which where still detectable at the six months follow up [30].
AAV-vector mediated gene transfer into murine hemopoietic cells was demonstrated by the conference of G418 resistance to murine in vitro colony forming units (CFU) following infection with a recombinant AAV-vector carrying the neo.sup.R -gene [32,33]. The presence of the vector in the progeny of CFU-GM (colony forming units-Granulocyte Macrophage) and BFU-E (burst forming units-Erythrocyte) was verified by means of PCR (Polymerase Chain Reaction). The efficiency of gene transfer varied between 0.5% and 15% [33]. Efficient gene delivery (up to 80%) into human hemopoietic progenitors and human CD34.sup.+ cells with AAV-neo.sup.R vectors has also been reported [34,35,36,37]. These studies demonstrated that rAAV vectors were able to deliver their DNA to the nucleus of the hemopoietic progenitor cells that can be cultured in vitro. Though delivery of the vector DNA to the nucleus of cells demonstrates the presence of a functional virus receptor on the surface of the target cells, delivery of rAAV to the nucleus of cells is not directly related to the integration of that DNA into the host cell genome (discussed later and presented in table 2). Recombinant adeno-associated virus DNA present as an episome in the cells is known to refrain from integration into the host cell genome in non-dividing tissue culture cells [38]. Integration of rAAV in CD34.sup.+ cells and in vitro growing colonies (CFU-C) was demonstrated in 1996 by Fischer-Adams et al. [59]. Stable transduction of P-PHSC is neither taught nor suggested in any of these prior art documents, however. None of the above mentioned studies discloses delivery and integration of rAAV to P-PHSC, the only relevant hemopoietic cell type for long term persistence of transduced cells in vivo.
We are developing rAAV gene transfer into P-PHSC for the treatment of .beta.-thalassemia and Sickle cell anemia. Both diseases severely affect the function of erythrocytes in these patients. .beta.-thalassemic erythrocytes contain insufficient .beta.-globin chains, whereas mutant .beta.-globin chains are made in sickle cell anemia (for review see [39]). Both diseases severely affect erythrocyte function which can be alleviated by persistent .gamma.-globin gene expression in the adult patient in which case fetal hemoglobin is formed [40]. Both inherited diseases are recessive in nature which indicates that one functional intact copy of the adult .beta.-globin gene is sufficient to ameliorate the phenotype.
Globin abnormalities were discarded as targets for gene therapy attempts in the early days of gene therapy research. This was largely due to the extremely complicated expression patterns of globin-like genes [41]. Globin-synthesis is highly regulated during development and confined to cells of the erythroid lineage. Furthermore, the expression of .alpha.- and .beta.-globin like chains is regulated such that they are maintained at a 1 to 1 ratio in the cell. Such careful control of gene expression is not easily obtained. Expression vectors carrying the human .beta.-globin gene with its promoter and local enhancer elements can direct erythroid specific globin RNA expression [42]. However, typically, the levels are less than 1% of the endogenous globin RNA.
Recently, sequences 50-60 kb upstream of the .beta.-globin gene were discovered that direct the high level, tissue specific, copy number dependent and position independent expression of the .beta.-globin gene [43]. This region, designated the Locus Control Region (LCR), is characterized by four strong erythroid-specific DNaseI hypersensitive sites (HS1-4) [44]. Fine-mapping of the active sequences in the LCR identified four fragments of .+-.400 bp in length that each coincide with one HS site. Walsh et al incorporated a marked .gamma.-globin gene and the core fragment of HS2 together with the neo.sup.R gene into an AAV-vector [20]. Infected and G418 selected pools and clones of K562 cells produced the marked .gamma.-globin RNA to 50-85% compared to the normal level expressed by one endogenous .gamma.-globin gene [20,45]. A drawback of this vector is that the .gamma.-globin gene and promoter used in these studies are specific for expression in fetal erythroid tissue and, thus, not ideal for use as a therapeutic agent in adult humans, tInorporation of .beta.-LCR sites 1, 2, 3 and 4 in a vector containing the adult specific human .beta.-globin gene resulted in a very high regulated expression in MEL (murine erythroleukemia) cells, the best in vitro marker cell line for regulated erythroid expression in adult tissue [46]. The present invention describes the use of this and similar vectors in the transduction of P-PHSC.
The term "infectious particles" is used herein to refer to AAV particles that can deliver their packaged DNA to the nucleus of cells and replicate in the presence of adenovirus and wtAAV.
The term "transducing particles" is used herein to refer to AAV particles that can deliver their packaged DNA to the nucleus of target cells where the packaged DNA is released and integrates into the chromosomal DNA of the target cells.